In search of the mechanism of photoresponses in Chlamydomonas reinhardtii: Behavioral and structural studies

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Chlamydomonas reinhardtii exhibit phototaxis and photophobic "stop" responses to temporal shifts in light intensity. This green algae contains an eyespot, an organelle thought to be involved in photoresponses. Strains CC-2359 and FN68 are found to be devoid of photophobic responses to photostimuli over a wide range of light intensities. No eyespot is seen in either mutant strain under the light microscope. Electron microscopy studies show that the eyespot in CC-2359 is absent, while other organelles are evidently unaffected. FN68 cells contain an eyespot which is altered soley with respect to the pigment granule content as compared to the structure found in wild type cells. Photophobic responses of both mutants are restored by exogenous addition of all-trans retinal and these responses are the same in extent and duration as the wt response. Therefore, the eyespot is not necessary for the photophobic response in Chlamydomonas; it is not the locus of reconstitution of this response, and it is not critical for the appropriate assembly and function of the photophobic response receptor in the membrane. Other laboratories report differences in phototactic behavior between CC-2359 and FN68. The data presented here indicate that an eyespot probably increases the stimulus differential received by a properly oriented cell, so that cells without an eyespot receive a weaker stimulus and are unable to perform the entire range of phototactic responses.;We have combined computer-based cell-tracking and motion analysis with retinal isomer and retinal analog reconstitution of CC-2359 and FN68 to investigate properties of the photophobic response receptor. Most rapid and most complete reconstitution is obtained with all-trans isomer of retinal. An analog locked by a carbon bridge in a 6-s-trans conformation reconstitutes whereas the corresponding 6-s-cis locked analog does not. Retinal analogs prevented from isomerization around the 13-14 double bond do not restore the response, but enter the chromophore binding pocket as evidenced by their inhibition of all-trans retinal regeneration of the response. Results of competition experiments between all-trans and each of the 13-locked analogs fit a model in which each chromophore exhibits reversible binding to the photoreceptor apoprotein. The chromophore requirement for the trans configuration and 6-s-trans conformation, and the lack of signaling function from analogs locked at the 13 position, are characteristic of archaebacterial rhodopsins, rather than the previously studied eukaryotic rhodopsins (i.e. visual pigments).